P.298 - Development of a CRISPR-based gene therapy approach targeting the large exon 45-exon 55 mutation hotspot in DMD gene

Abstract

Gene replacement and exon skipping therapies for Duchenne muscular dystrophy (DMD) showed successful improvements in preclinical studies. However, results obtained in recent clinical trials called for the development of new therapeutic strategies that would restore a full-length or nearly full-length dystrophin expression, provide long-term correction and answer challenges regarding body-wide targeting. Dmd gene editing approaches hold great promise and indeed have been shown to successfully excise exon 23 and exons 52–53 in vivo, leading to histopathological improvement. Targeting the larger exon 45-exon 55 (Ex45-Ex55) mutation hotspot would account for about 60% of the DMD patients. A proof-of-concept study validated this approach in iPS cells. We are developing a CRISPR-based gene therapy approach targeting the Dmd Ex45-Ex55 region to restore reading-frame and protein expression in vivo. Both Streptococcus pyogenes (sp) and Staphylococcus aureus (sa) Cas9 endonucleases are used to edit Dmd gene following AAV transfer. First, gRNAs were designed and validated in vitro. Selected gRNAs have been injected either in mouse models with muscle-specific Cas9 expression or in wild-type mice with spCas9 or saCas9 as single or dual AAV vectors. Cas9 expression was confirmed at the RNA and protein levels. Nuclear localization and transduction efficiency was checked by immuno-histo-fluorescence (IHF). Endonuclease activity at the targeted loci and Ex45-Ex55 inversion or deletion events are being investigated at the DNA and RNA levels by PCR, qPCR and ddPCR.